Name: Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors (Video) | JoVE Journal
Uploaded: 2018-12-14T13:00:00.000+00:00
Duration: 11 min 46 s
Description: Learn direct lineage reprogramming to convert mouse fibroblasts into erythroid progenitors for studying transcriptional regulation. This method enables efficient manipulation of cell fate and supports transfusion medicine research.

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5 minutes

We thank Evelyn Wang and Gregory Hyde (Whitehead Institute, Cambridge, MA) for cloning and Harvey Lodish (Whitehead Institute) for providing many of the plasmids used for generating the retroviral library. We thank Kavitha Siva and Sofie Singbrant (Department of Molecular Medicine and Gene Therapy, Lund University), G&#246;ran Karlsson and Shamit Soneji (Department of Molecular Hematology, Lund University) for their roles in description of iEP production. We would also like to acknowledge and thank Julian Pulecio (Centre of Regenerative Medicine, Barcelona Biomedical Research Park), Violeta Rayon-Estrada (The Rockefeller University, New York), Carl Walkley (St. Vincent's Institute of Medical Research and Department of Medicine, St Vincent's Hospital, University of Melbourne), &#193;ngel Raya (Catalan Institution for Research and Advanced Studies, Barcelona), and Vijay G. Sankaran (Broad Institute of the Massachusetts Institute of Technology and Harvard, Cambridge) for their previous contributions to this work. This work was supported by the Ragnar S&#246;derberg Foundation (to J.F.); the Swedish Research Council (to J.F.); Stiftelsen Olle Engkvist Byggm&#228;stare (to J.F.); the Swedish Foundation for Strategic Research (to J.F.); &#197;ke Wiberg's Foundation (to J.F.); a Marie Curie integration grant (to J.F.).

1. Establishment and Maintenance of Primary Mouse Tail Tip Fibroblast Cultures
<ol>
	<li>Prepare gelatin-coated dishes (recommend a 10 cm dish for one tail) by covering the surface with 0.1% gelatin and incubating the dishes for approximately 20 min at 37 &#176;C. Aspirate the gelatin solution from the dish and allow it to dry for at least 2 h.</li>
	<li>Euthanize the mice by cervical dislocation. Remove the tail with scissors, cutting at the base of the tail. Put the tail in Dulbecco&#39;s phosphate-buffered saline (DPBS) with 2% fetal bovine serum (FBS) until ready to use. 
	NOTE: For the best results, tails should be taken from mice around 6 to 8 weeks of age. Tails can be taken from mice older than 8 weeks of age, however, as the mice age, the proliferation capability of the fibroblasts and the efficiency of reprogramming decrease11.</li>
	<li>Perform all subsequent steps of this protocol in a tissue culture hood under sterile conditions. Prepare a diluted trypsin solution of 0.02% trypsin-EDTA in DPBS and add 5 mL into an uncoated 10 cm dish.</li>
	<li>Wash the tail, first in 70% ethanol, then in DPBS. In a dish, place the tail flat and use forceps to hold it in place. Make an incision on the tail along its longitudinal axis from the base to the tip.</li>
	<li>Grasp the tail with one pair of forceps and hold it vertically. Using a second pair of forceps, grip the skin beside the incision at the base of the tail and peel it back. Do this at both sides of the incision until the skin can be peeled off by pulling downwards toward the tip of the tail.</li>
	<li>Hold the peeled tail with forceps over the dish containing the trypsin solution and cut the tail into approximately 1 cm long pieces. With the tail pieces in the trypsin solution, use scissors to fragment the pieces into smaller pieces. Incubate the tail pieces in the trypsin solution at 37 &#176;C for 10 min. 
	NOTE: The smaller the pieces are preferred so as to provide each piece a high surface area to volume ratio.</li>
	<li>Quench the trypsin using 2 volumes of fibroblast expansion (FEX) medium (high-glucose Dulbecco Modified Eagle Medium (DMEM) with 15% FBS, 2 mM L-glutamine, Non-essential amino acids (NEAA) and 100 U/mL Penicillin/Streptomycin).</li>
	<li>Collect entire contents of the dish into a 50 mL tube and centrifuge at 350 &#215; g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend the tail fragments in 10 mL of fresh FEX medium.</li>
	<li>Transfer tail fragments in medium to a gelatin-coated dish and incubate at 37 &#176;C in 5% CO2 and 4% O2, adding fresh FEX medium every 2 days. 
	NOTE: After five to seven days, tail fragments have attached at the bottom of the dish and fibroblasts can be seen moving away from them.</li>
	<li>Once clusters of fibroblasts are spotted, gently shake the dish to dislodge tail pieces and aspirate the medium and all the bone fragments leaving the fibroblasts attached to the plate. Add new FEX medium and culture the fibroblasts until confluent.</li>
	<li>To ensure no contamination of fibroblasts by hematopoietic progenitors, dissociate the cells from the plate using 1x trypsin-EDTA for 5 mins and collect cells. Deplete for cells expressing hematopoietic markers (CD117, CD5, CD45R (B220), CD11b, Anti-Gr-1 (Ly-6G/C), 7-4, and Ter-119) using a magnetic separation system10.</li>
</ol>
2. Retrovirus Production
<ol>
	<li>Seed retroviral packaging cells at approximately 2.5 &#215; 104 cells/cm2 (2.0 &#215; 106 cells for a 10 cm dish) on a tissue culture-treated (by vacuum-gas plasma) dish and culture overnight in high-glucose DMEM with 10% FBS, 10 mM Sodium Pyruvate, and 100 U/mL Penicillin/Streptomycin at 37 &#176;C and 5% CO2.</li>
	<li>On the following morning, change the medium to DMEM with no additives using half the volume used to culture overnight (5 mL for a 10 cm dish). In the afternoon, check that the cells are 70-80% confluent and begin the transfection.</li>
	<li>For each reprogramming factor, Gata1, Tal1, Lmo2, and c-Myc, prepare a 2:1 mixture of the expression vector (pMX) and helper vector (containing gag and pol genes). For a 10 cm dish of fibroblasts, use 6 &#181;g of expression vector and 3 &#181;g of helper vector in a final volume of 100 &#181;L in DMEM medium.</li>
	<li>For each reprogramming factor, prepare 300 &#181;L of room temperature (RT) DMEM in a sterile polystyrene tube and carefully add 27 &#181;L of commercial transfection reagent. 
	NOTE: The transfection reagent should be brought to RT before use and must be added directly into the medium as the electrostatic properties of the compound can make it stick to the plastic wall of the tube.</li>
	<li>Add the plasmid mix into the transfection reagent-containing tube, vortex briefly and incubate the transfection reagent-DNA mixture for 15 min at RT. Briefly vortex the mixture and add it dropwise to the retroviral packaging cells so that the transfection reagent-DNA mix is evenly spread over the culture and incubate at 37 &#176;C overnight.</li>
	<li>24 h after transfection, change the medium to DMEM with 20% FBS and 100 U/mL Penicillin/Streptomycin. 48 h after transfection, collect the supernatant and filter it through a 0.22 &#181;m pore-size syringe filter. 
	NOTE: Viral supernatants can be frozen to -80 &#176;C and kept until required, although transduction is more efficient if fresh viral supernatant is used.</li>
</ol>
3. GTLM transduction and iEP harvest
<ol>
	<li>Seed the tail tip fibroblasts at 1 &#215; 104 cells/cm2 on 0.1% gelatin pre-coated dishes in FEX medium and incubate at 37 &#176;C for 24 h.</li>
	<li>The following day, prepare a transduction mixture as follows.
	<ol>
		<li>Add 1 volume of viral supernatant for each reprogramming factor supplemented with 4 &#181;g/mL of retroviral infection reagent (40%) to 6 volumes of FEX medium (60%).</li>
		<li>For a 10 cm dish of fibroblasts, add 1 mL of each viral supernatant (4 &#215; viruses = 4 mL) supplemented with 4 &#181;g/mL of retroviral infection reagent to 6 mL of FEX medium, giving a total of 10 mL transduction mixture.</li>
	</ol>
	</li>
	<li>Aspirate FEX medium from the fibroblast culture and replace it with the transduction mixture. Incubate the transduction for 4 h at 37 &#176;C in hypoxic conditions (5% CO2 and 4% O2).</li>
	<li>Aspirate the transduction mixture and replace it with fresh reprogramming medium (Serum-free Expansion medium (SFEM), 100 U/mLPenicillin/Streptomycin, 100 ng/mL murine Stem Cell Factor (mSCF), 10 ng/mL murine interleukin-3 (IL3), 2 U/mL human recombinant Erythropoietin (hrEPO), and 100 nM Dexamethasone).</li>
	<li>Incubate for 8 daysat 37 &#176;C in hypoxic conditions, adding fresh reprogramming medium every 2 days. After five to eight days, successful reprogramming will yield clusters of cells that have detached from the plate.</li>
	<li>To collect reprogrammed cells for analysis, gently pipette up and down to harvest them directly from the dish. To harvest untransduced fibroblasts for comparison, dissociate the cells from the plate using 1x trypsin-EDTA and collect.</li>
</ol>

<ol><li>Sender, R., Fuchs, S., Milo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Are+We+Really+Vastly+Outnumbered%3F+Revisiting+the+Ratio+of+Bacterial+to+Host+Cells+in+Humans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Are We Really Vastly Outnumbered? Revisiting the Ratio of Bacterial to Host Cells in Humans.</a> Cell. 164 (3), 337-340 (2016).</li>
<li>Lujan, E., Chanda, S., Ahlenius, H., Sudhof, T. C., Wernig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+conversion+of+mouse+fibroblasts+to+self-renewing%2C+tripotent+neural+precursor+cells%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (7), 2527-2532 (2012).</li>
<li>Szabo, E., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/21057492">Direct conversion of human fibroblasts to multilineage blood progenitors.</a> Nature. 468 (7323), 521-526 (2010).</li>
<li>Pereira, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+a+hemogenic+program+in+mouse+fibroblasts%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Induction of a hemogenic program in mouse fibroblasts.</a> Cell Stem Cell. 13 (2), 205-218 (2013).</li>
<li>Batta, K., Florkowska, M., Kouskoff, V., Lacaud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+reprogramming+of+murine+fibroblasts+to+hematopoietic+progenitor+cells%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells.</a> Cell Reports. 9 (5), 1871-1884 (2014).</li>
<li>Yu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reprogramming+fibroblasts+into+bipotential+hepatic+stem+cells+by+defined+factors%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.</a> Cell Stem Cell. 13 (3), 328-340 (2013).</li>
<li>Hendry, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+transcriptional+reprogramming+of+adult+cells+to+embryonic+nephron+progenitors%5BTitle%5D)+AND+%22Journal+of+the+American+Society+of+Nephrology%22%5BJournal%5D)">Direct transcriptional reprogramming of adult cells to embryonic nephron progenitors.</a> Journal of the American Society of Nephrology. 24 (9), 1424-1434 (2013).</li>
<li>Vierbuchen, T., Wernig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+lineage+conversions%3A+unnatural+but+useful%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Direct lineage conversions: unnatural but useful.</a> Nature Biotechnology. 29 (10), 892-907 (2011).</li>
<li>Capellera-Garcia, S., Flygare, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+lineage+reprogramming%3A+a+useful+addition+to+the+blood+cell+research+toolbox%5BTitle%5D)+AND+%22Expert+Review+of+Hematology%22%5BJournal%5D)">Direct lineage reprogramming: a useful addition to the blood cell research toolbox.</a> Expert Review of Hematology. 10 (2), 107-109 (2017).</li>
<li>Capellera-Garcia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+Minimal+Factors+Required+for+Erythropoiesis+through+Direct+Lineage+Conversion%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Defining the Minimal Factors Required for Erythropoiesis through Direct Lineage Conversion.</a> Cell Reports. 15 (11), 2550-2562 (2016).</li>
<li>Mahmoudi, S., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aging+and+reprogramming%3A+a+two-way+street%5BTitle%5D)+AND+%22Current+Opinions+in+Cellular+Biology%22%5BJournal%5D)">Aging and reprogramming: a two-way street.</a> Current Opinions in Cellular Biology. 24 (6), 744-756 (2012).</li>
<li>Singbrant, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Erythropoietin+couples+erythropoiesis%2C+B-lymphopoiesis%2C+and+bone+homeostasis+within+the+bone+marrow+microenvironment%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Erythropoietin couples erythropoiesis, B-lymphopoiesis, and bone homeostasis within the bone marrow microenvironment.</a> Blood. 117 (21), 5631-5642 (2011).</li>
<li>Heinrich, A. C., Pelanda, R., Klingmuller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+for+visualization+and+conditional+mutations+in+the+erythroid+lineage%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">A mouse model for visualization and conditional mutations in the erythroid lineage.</a> Blood. 104 (3), 659-666 (2004).</li>
<li>Gautier, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene-expression+profiles+and+transcriptional+regulatory+pathways+that+underlie+the+identity+and+diversity+of+mouse+tissue+macrophages%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages.</a> Nature Immunology. 13 (11), 1118-1128 (2012).</li>
<li>Psaila, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+profiling+of+human+megakaryocyte-erythroid+progenitors+identifies+distinct+megakaryocyte+and+erythroid+differentiation+pathways%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways.</a> Genome Biology. 17 (1), 83(2016).</li>
<li>Pulecio, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+Conversion+of+Fibroblasts+to+Megakaryocyte+Progenitors%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Direct Conversion of Fibroblasts to Megakaryocyte Progenitors.</a> Cell Reports. 17 (3), 671-683 (2016).</li>
<li>Kurita, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+immortalized+human+erythroid+progenitor+cell+lines+able+to+produce+enucleated+red+blood+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.</a> PLoS One. 8 (3), 59890(2013).</li>
</ol>

The authors have no conflicts of interest to report.

Overexpression of a four-factor cocktail, GATA1,&#160;TAL1,&#160;LMO2,&#160;and c-MYC (GTLM), is sufficient to reprogram murine and human fibroblasts directly to iEPs10. The reprogrammed erythroid cells greatly resembled bona fide erythroid progenitors in terms of morphology, phenotype, gene expression, and colony-forming ability. This finding corroborates the rationale of using direct ...

Red blood cells (RBCs) are essential in all vertebrates and make up 84% of all cells of human bodies1. From embryonic to adult life, our health is highly dependent on exact regulation of RBC homeostasis. The ongoing production of mature RBCs throughout development into adulthood is known as erythropoiesis. A major challenge in erythropoiesis research is to define the master regulators that orchestrate RBC development and the switch between primitive and definitive erythropoiesis. Direct lineage reprogramming of erythroid progenitors presents an opportunity to further understand erythroid development in vivo.
Direct lineage reprogramming (DLR), also known as transdifferentiation, is the process of reprogramming one cell type directly into another, bypassing pluripotent and multipotent progenitor stages. DLR has thus far been used to produce numerous cell types including neural2, hematopoietic3,4,5, hepatic6 and nephrotic7, progenitor cells. For developmental biologists, DLR has become an important tool for interrogating aspects of lineage commitment and terminal differentiation processes8,9. DLR can complement and partially replace in vivo studies for understanding mechanisms of cell fate determining factors during development. The DLR protocol for reprogramming to erythroid progenitors described in this paper provides the field a complimentary method for developmental studies of erythropoiesis.
We have previously demonstrated that overexpression of a four-factor cocktail, GATA1, TAL1, LMO2, and c-MYC (GTLM), is sufficient to reprogram both murine and human fibroblasts directly to induced erythroid progenitors (iEPs)10. The GTLM-reprogrammed erythroid cells greatly resemble bona fide primitive erythroid progenitors in terms of morphology, phenotype, and gene expression10. Thus iEPs have limited proliferation capacity and mature to nucleated erythrocytes similar to those transiently produced in the early embryo before onset of definitive erythropoiesis. By making changes in the reprogramming conditions (e.g., point mutations in reprogramming factors or addition of other factors), one can understand how this leads to changes in erythroid development and differentiation. We have for example shown that addition of Klf1 or Myb to the GTLM cocktail changes the globin expression pattern from predominantly embryonic (primitive) to mainly adult (definitive). This finding corroborates the validity of using DLR as a tool for defining developmental factors in erythropoiesis.
Here, we outline the process of generating iEPs from mouse tail tip fibroblasts (TTF). In our representative results, we performed the reprogramming on fibroblasts from the erythroid lineage-tracing mice (Epor-Cre R26-eYFP) which express the yellow fluorescent protein (eYFP) from the Rosa26 locus in all cells that have expressed the erythropoietin receptor, allowing easy visualization of commitment to the erythroid lineage. Using this method, YFP positive (EpoR+) cells are present as early as five days after transduction. This protocol, therefore, offers a quick and robust technique for the generation of erythroid progenitors in vitro.

<table><tbody><tr><td>DMEM without sodium pyruvate</td><td>GE Life Sciences</td><td>SH30022.01</td><td>Culturing media for PhGP cells</td></tr><tr><td>DMEM with sodium pyruvate</td><td>GE Life Sciences</td><td>SH30243.01</td><td>Culturing media for tail tip fibroblasts</td></tr><tr><td>StemSpan Serum Free Expansion Media (SFEM)</td><td>Stem Cell Technologies</td><td>9650</td><td>Reprogramming media&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum HyClone</td><td>GE Life Sciences</td><td>SH30071.03HI</td><td>Growth factor</td></tr><tr><td>Penicillin/Streptomycin HyClone (100x)</td><td>Ge Life Sciences</td><td>SV30010</td><td>Antiboiotic</td></tr><tr><td>Non-Essential Amino Acids (100x)</td><td>Thermo Fisher</td><td>11140050</td><td>SNL media is suppplemented with this</td></tr><tr><td>Trypsin HyClone (1x)</td><td>GE Life Sciences</td><td>SH30042.01</td><td>Cell dissociation agent</td></tr><tr><td>Murine Stem Cell Factor (mSCF)</td><td>Peprotech</td><td>250-03</td><td>Added to reprogramming media</td></tr><tr><td>Recombinant Murine Il-3</td><td>Peprotech</td><td>213-13</td><td>Added to reprogramming media</td></tr><tr><td>human recombinant erythropoietin (hrEPO)</td><td>Peprotech</td><td>100-64</td><td>Added to reprogramming media</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>50-02-2&amp;nbsp;&amp;nbsp;</td><td>Added to reprogramming media</td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>9000-70-8&amp;nbsp;</td><td>Dissovled in dH2O and used for coating plates. Ensure sterility before use.</td></tr><tr><td>Blasticidin S hydrochloride</td><td>Sigma</td><td>3/9/3513</td><td>Selection antibiotic. Affects both pro- and eukaryotic cells</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>Ge Life Sciences</td><td>SH30850.03</td><td>Used for washing steps</td></tr><tr><td>Polybrene</td><td>Merck</td><td>TR-1003-G</td><td>Infection / Transfection&amp;nbsp; Reagent</td></tr><tr><td>FuGENE HD Transfection Reagent</td><td>Promega</td><td>E2311</td><td>Transfection Reagent for PhGP cell line</td></tr><tr><td>Millex-GP Syringe Filter Unit 0.22 &amp;micro;m</td><td>Merck</td><td>SLGP033RS</td><td>Used for filtering virus supernatant</td></tr><tr><td>BD Emerald Hypodermic Syringe</td><td>Becton Dickinson</td><td>SKU: 307736</td><td>Used for filtering virus supernatant</td></tr><tr><td>100 mm Culture Dish</td><td>Corning</td><td>430167</td><td>Cell culture</td></tr><tr><td>6-well plate</td><td>Falcon</td><td>10799541</td><td>Cell culture</td></tr><tr><td>Jeweler Forceps #5</td><td>Sklar</td><td>66-7642</td><td>Used for handling small tail fragments</td></tr><tr><td>Sklarlite Iris Scissors</td><td>Sklar</td><td>23-1149</td><td>Used for cutting the tail into small pieces</td></tr><tr><td>Lineage Cell Depletion Kit, mouse</td><td>Miltenyi Biotec</td><td>130-090-858</td><td>For depletion of hematopoietic cells in fibroblast cultures</td></tr><tr><td>CD117 MicroBeads, mouse</td><td>Miltenyi Biotec</td><td>130-091-224</td><td>For depletion of hematopoietic cells in fibroblast cultures</td></tr><tr><td>PheonixGP cells</td><td>ATCC</td><td>CRL-3215</td><td>retroviral packaging cell line</td></tr><tr><td>EcoPAC vector (pCL-Eco)&amp;nbsp;</td><td>Novus Biologicals</td><td>NBP2-29540</td><td>retroviral helper vector containing gag and pol genes</td></tr><tr><td>pMX-Gata1</td><td>Cloned in-lab</td><td></td><td></td></tr><tr><td>pMX-Tal1</td><td>Cloned in-lab</td><td></td><td></td></tr><tr><td>pMX-Lmo2</td><td>Cloned in-lab</td><td></td><td></td></tr><tr><td>pMX-cMyc</td><td>Cloned in-lab</td><td></td><td></td></tr><tr><td>CellSens Standard 1.6 software&amp;nbsp;</td><td></td><td></td><td>Cytospin analysis software</td></tr></tbody></table>

direct lineage reprogramming of adult mouse fibroblast to erythroid progenitors

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present a reproducible protocol for the production of iEPs from adult fibroblasts using transcription factor-driven DLR. We evaluate the cell reprogramming using flow cytometry, colony-forming assays, and gene expression analysis. In order to assist in the visualization of the conversion to erythroid cell fate, we performed the reprogramming on fibroblasts from the erythroid lineage-tracing mice (Epor-Cre R26-eYFP) which express the yellow fluorescent protein (eY...

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

direct-lineage-reprogramming-adult-mouse-fibroblast-to-erythroid

Research

JoVE Journal

Developmental Biology

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Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-β signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

Learn direct lineage reprogramming to convert mouse fibroblasts into erythroid progenitors for studying transcriptional regulation. This method enables efficient manipulation of cell fate and supports transfusion medicine research.

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

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